Induction of Adenosine A_1 Receptor Expression by Pertussis Toxin via an Adenosine 5'-Diphosphate Ribosylation-Independent Pathway

摘要

Pertussis toxin ADP ribosylates G_1 and G_0 transducing proteins and functionally uncouples adenosine A_1 receptor (A_1AR) from its effectors.We hypothesized that this loss in receptor coupling could lead to de novo A_1AR synthesis by the cell in a futile attempt to re-establish normal receptor function.To test this hypothesis,we used hamster ductus deferens tumor (DDT_1 MF-2) cells,a cell culture model for studying A_1AR,and showed that pertussis toxin (100 ng/ml) produced a time-dependent loss in A_1AR-G_i interaction and abolished A_1AR activation of extracellular signal-regulated kinase 1/2.Interestingly,pertussis toxin increased the expression of A_1AR,as measured by real-time polymerase chain reaction,immunocytochemistry,and [~3H]cyclopentyl-1,3-dipropylxanthine (DPCPX) binding,suggesting a compensatory response to G_i protein inactivation.DDT_1 MF-2 cells exposed to pertussis toxin demonstrated nuclear factor kappa B (NF-kappa B) activation within 30 min of exposure,a time point that preceded the loss of function of the A_1AR.Inhibition of NF-kappa B attenuated the increase in A_1AR induced by pertussis toxin.Cells exposed to B-oligomer subunit of pertussis toxin,devoid of significant ADP ribosyltransferase activity,showed increased A_1AR protein expression,preceded by activation of NF-kappa B.B-Oligomer increased intracellular Ca~(2+) in DDT_1 MF-2 cells.Chelation of intracellular Ca~(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or inhibition of protein kinase C (PKC) with bisindolylmaleimide hydrochlo-ride reduced the activation of NF-kappa B and [~3H]DPCPX binding.We conclude that pertussis toxin promotes de novo A_1AR synthesis by activating NF-kappa B through an ADP ribosylation-independent mechanism involving intracellular Ca~(2+) release and PKC activation.